CERTIFICATE OF TRANSLATION I, Kazuo YAMASAKI, Patent Attorney of c/o NAKAMURA & PARTNERS, Shin-Tokyo Building, No. 3-1, Marunouchi 3-chome, Chiyoda-Ku, Tokyo, JAPAN, hereby certify that to the best of my knowledge and belief the attached English translation is a true translation, made by me and for which I accept responsibility, of the PCT application number PCT/JP2005/020195 filed on November 2, 2005 in the name of ASAHI BREWERIES, LTD. April 18, 2007 Kazuo YAMASAKI Patent Attorney English translation of PCr/JP2005/020195 Y1M0838 1/5 o R AM 0-1 BmE% 0-2 IfiN 0-3 ('.lfl) 0-4 04 PCT/RO/101 0-4-1 t e o ar=t. JPO-PAS 0330 0-5 Zt 0-6 aIttrnm A ($I (RO/JP) 0-7 xt Y1M0838 II UiMA l-I :mm:efLt ±is $ A' 7 (applicant only) 11-2 <®R TLt * ~ :-~( CO MT)M (all designated States except US) -4 a 7 #1 t" l-4en Name. ASAHI BREWER IES, LTD. II-s5ja ar 1048323 A-, Z 3TH 79 1 4 Il-Sen Address: 7-1, Kyobashi 3-chome, Chuo-ku, Tokyo 1048323 Japan 11-6 I% (El4) F39 gJP 11-7 (@?]4 9 gJP 11-11 moet- 000000055 Title of the Invention MEMBRANE FILTER FOR MICROBE DETECTION Y1M0838 2/5 11-1-1 : _- 04im LtAM A , @A (applicant and inventor) 1-1-2 to$UO TO A* d$90.4, I)l (US only) Ill-1-4ja IMA44)j P lII-1-4en Name (LAST, First): MOTOYAMA Yasuo .l-1-Sja a-t 1308602 1-3?E [I m 4 1 TI 2 3# 1 7-1-t E-1 In-1-5en Address: C/o RESEARCH & DEVELOPMENT PLANNING DEPARTMENT, ASAHI BREWERIES, LTD., 23-1, Azumabashi 1-chome, Sumida-ku, Tokyo 1308602 Japan 111-1-6 E(l) JP 111-1-7 (fr(4) E JP 111-2 A 111-2-1 )1 ELtt i , ARU 9 (applicant and inventor) 11-2-2 to m.) 05NIzD IM *M)- (US only) 1I-2-4ja Ft (Ott) ? itf ll-2-4en Name (LAST, First): YASUHARA Takaomi 111-2-5jia VC: 1308602 I T H42 3-# 1T 2 301 7+t "-t0-l, l11-2-5en Address: C/o RESEARCH & DEVELOPMENT PLANNING DEPARTMENT, ASAHI BREWERIES, LTD., 23-1, Azumabashi 1-chome, Sumida-ku, Tokyo 1308602 Japan 111-2-6 MOM9% ) E(9 JP 111-2-7 flWr ([E4) i* 9 J Y1M0838 3/5 IV-1 REAlth20RW, bho~t4 eoimunfm R A (agent) IV-1lia RA(RSt) ga MI IV-1len Name (LAST, First): KUMAKURA Yoshi o IV--2ja ATA 1008355 + FR 1lA) A 3 T R 3 1 R. E)11, IV- 1-2enAddress: NAKAMURA & PARTNERS, Shin-Tokyo Bldg., 3-1, Maru nouchi 3-chome, Chiyoda-ku, Tokyo 1008355 Japan IV- 1-3 a * 03-3211-8741 IV- 1-4 7-, 03-3214-6358 IV-1-6 WTA # 100082005 Iv-2 _Etof]A^ *HR IAk181MCto2b-CE 1W4 1 I A I (additional agent(s) with the same address as first named agent) IV-2-lIja L tNJll a (100084009); Eim X (100084663) A# WA(100093300); TI & 9- (100114007): IJht -; (100119013) IV-2-len Name(s) G06AWA Nobuo (100084009) ; HAKODA Atsushi (100084663); ASAI Kenji (100093300); HIRAYAMA Koji (100114007); YAMASAKI Kazuo(100119013) v M19 V-1 LOjffoitDBilil 4.9(a)t iDamacA, i ,-Jt L6:T--PCTOiff )UMtWL, l, 567 4660o~ilkte. At/IEjf vi-11 M 20044 11,A 02l (02. 11.2004) VI-1-2 BM94 2004-319479 vl-1-3 Mt E I*V jp VI-2 #%Eli t vil-1 ustuP_ aME. M(ISA) B_(ISA/JP) Y1 M0838 4/5 VIII-2 ~ ~ ~ ~ Ep _q__ __ __-_ __ VIII-4 (3~ VIII-5 TJ~ IX-1 om1 I]I- 5/ IX-2 RAa 5/ IX-3 1/ IX-4 !1/ JX-5 19E 3 / IX-7 e .ff+5__ _ _ _ _ _ _ _ _ _ _ IX-8 / IX-17 PCT-SAFE laTI IX-191 WM~ T-,t51 )t4 IX-2 I )WA , = X-2 I5A /100084005/ X-1-2 JF-O)"Z X-2-3 40_____________________________ X-2 dtISK /100084663/ X-2-1 P~V(Jj G) X-2-2 U) X-2-3 ____________________________ __ X-3 /100084600/ X-4-2 -tt ) X-4-3 ~I YIM0838 5/5 X-6 tHIiA, Y~ /1 0011907/ X-5-2 7 , ) 1U X-5-3 MU_ _ _ _ _ __ _ _ _ _ _ _ __ _ _ _ _ _ _ X--2 74,O) 10-3 MW LIf 101 EWIL jr~ i~tt% __ ) A mII 01 11)f DE 10-2 19 r0 10-- 1 15L ! Y1M0838 0-11 0-2 T-T 0 )F3fIq 0-4 4*PCT/RO/101 (ftMIF) 0-4-1 ~If~t~.JPO-PAS 0330 0-9 Wm~~tA~$4 YIM0838 2 fffik7 -it L-J* AtR4 12 FT)*vO#§+ _______ J4+ (JPY) 12-1 it--*04 T 4>13000 12-2 Nil-T~ 4 S 97000 12-3 FL TO 4 (RTO30#11tC) ii 123200 12-4 30tk~;5#V~k 0 12-5 ~ 1Q#&4(X) 0 12-6 i2 0 12-7 il +i2 = 123200 12-12 fully electronic filing fee reduction R -26400 12-13 ~~4 )t(i-R) Ig 96800 12-17 ~ -*cg+(T+S+I+P) C> 1206800 12-19 AW A il k 9.: -T M I3174LL,0 12-20 ~TMDI2 -t EM MAWT (RO/JP) 12-20-1 L1I i z I -1, I 12-21 228497 12-22 F31 20054 11A~ 02B (02. 11.2005) DESCRIPTION MEMBRANE FILTER FOR MICROBE DETECTION 5 Technical Field The present invention relates to a membrane filter for the detection of microorganisms, which has a low background fluorescence intensity and whose surface is smooth. 10 Background Art There has been known a method which comprises the steps of filtering off microorganisms by the use of a membrane filter, staining the microorganisms trapped on the membrane filter with a fluorescent substance and then observing the microorganisms with a fluorescence microscope. However, this method has a 15 problem that the membrane filter per se also emits fluorescence, and accordingly the method can never detect microorganisms with a high sensitivity. Examples of methods for the observation of such microorganisms include a method in which they are observed with the naked eye and a method comprising taking the image of the microorganisms with a CCD camera. In both of these methods, however, 20 microorganisms cannot be detected with a high sensitivity in a case where it is difficult to discriminate between the background fluorescence and the fluorescence originating from the microorganisms concerned. To solve the foregoing problems, it is necessary that the membrane filter be stained with a black dye such as Irgalan Black to thus reduce the background 25 fluorescence intensity, or to use a commercially available membrane filter stained with a black dye in advance (such as one available from Whatman under the trade name of CycloblackTM). Although the use of such a previously stained membrane filter would certainly result in the achievement of a background fluorescence-reducing effect with respect to some specific wavelengths, the 1 background fluorescence emitted from the stained membrane filter is not always low enough over the entire fluorescent wavelength region. More specifically, a problem arises such that microorganisms cannot be stained with a combination of fluorescent dyes capable of emitting fluorescence of a variety of wavelengths 5 according to any conventional methods, and accordingly the testing methods which can be used are limited. On the other hand, the materials used for forming the membrane filter well adapted for the foregoing fluorescent staining are in general polycarbonates and polyesters, but these materials have a variety of problems in that they are soft 10 and quite susceptible to rolling, that the surface of the membrane filter is correspondingly quite easily wrinkled and that the position of the focus is shifted each time the observed field is changed, in a case of microscopic observation. Moreover, a problem further arises such that these materials can be handled only with great difficulty when picking up the membrane filter with a pincette. 15 Disclosure of the Invention Problems that the Invention is to Solve Accordingly, it is an object of the present invention to provide a membrane filter for the detection of microorganisms which has a low background 20 fluorescence intensity, which can be adapted the staining with a combination of fluorescent dyes capable of emitting fluorescence of various wavelengths and which is improved in handleability. Means for Solving the Problems 25 The inventors of the present invention have conducted various studies to accomplish the foregoing objects, and as a result have found that the foregoing problems can be efficiently solved by coating the surface of a membrane filter with a metal and have thus completed the present invention. Accordingly, the present invention herein provides a membrane filter for 2 the detection of microorganisms which comprises a polymer layer having a thickness in the range of from 10 to 500 g m and a metal layer which is formed on the foregoing polymer layer and which has a thickness in the range of from 0.1 nm to 1p m. 5 Moreover, the present invention likewise provides a method for the detection of microorganisms which comprises the step of detecting the microorganisms using a membrane filter for the detection of microorganisms as set forth in any one of claims 1 to 5. 10 Best Mode for Carrying Out the Invention The membrane filter for the detection of microorganisms according to the present invention comprises a polymer layer and a metal layer. The polymer constituting the polymer layer is not restricted to any particular one so long as it is a polymer material currently used for forming a 15 membrane filter and examples thereof include nitrocellulose, polycarbonate, polyester, polysulfone, polyfluoroethylene, polyethylene and polypropylene. Preferably used herein are polyesters and polycarbonates. The membrane filters prepared from polyesters, polycarbonates and the like comprise fine pores having a uniform size, and therefore they are quite suitably used for trapping bacteria 20 and for the observation of the trapped bacteria while staining the same with a fluorescent dye. The polymer layer may be formed according to any conventionally used method and it is also possible to use any commercially available membrane filter such as CycloblackTM (available from Whatman Corporation) and NucleporeTM 25 (available from Whatman Corporation) as the polymer layer for the membrane filter of the present invention. The thickness of the polymer layer preferably ranges from 10 to 500 u m and more preferably 20 to 30 p m. The metal constituting the metal layer is not likewise restricted to any 3 specific one and examples thereof include metals such as gold, silver, copper, zinc, aluminum, titanium, tantalum, chromium, iron, nickel, cobalt, lead and tin; or alloys of these metals (provided that the metal layer is not constituted by Au, Pt, Pd, and Pt-Pd). Preferably used herein is tin. The use of tin is particularly 5 effective, since tin is free of any toxicity to microorganisms and it makes the observation of bacteria after staining the same easy. The method for forming such a metal layer on the foregoing polymer layer is not limited to any specific one so long as it may allow the formation of a smooth film and specific examples thereof include the evaporation method, the sputtering 10 method and the CVD method. The thickness of the metal layer is preferably in the range of from 0.1 nm to 1 m and more preferably 10 to 100nm. If the thickness of the metal layer falls within the range specified above, the metal layer permits the realization of a membrane filter possessing a reduced background fluorescence intensity, a 15 smoother surface and excellent handleability. The fine pore size, 4, of the membrane filter according to the present invention is preferably in the range of from 0.1 to 10 , m, but the pore size is preferably selected depending on the size of each particular microbial cell used as a subject. 20 The density of the fine pores for the membrane filter is preferably in the range of from 105 to 108 pores/m 2 . Such a membrane filter shows a tendency that the higher the fine pore density, the better the filtering capacity of the filter, but the lower the strength of the same. Accordingly, one should select a membrane filter having a fine pore size and a fine pore density which takes these 25 characteristic properties into account. The membrane filter according to the present invention may likewise be prepared by first forming a metal layer on a polymer layer and then forming fine pores on the resulting assembly or it may be prepared by forming a metal layer on a polymer layer on which fine pores have been formed in advance. 4 The method for forming fine pores may be any one of those currently used and, for instance, includes a method comprising the steps of irradiating a polymer layer with charged particles or neutrons to thus form trajectories thereof through the layer and then chemically etching the trajectories. It would be better to form 5 such fine pores in such a manner that they have a uniform pore size and cylindrical pore channels formed through the film which are arranged in a direction perpendicular to the surface thereof. In this connection, when forming the metal layer according to, for instance, the evaporation technique, the fine pore size of the membrane filter according to the present invention is smaller than that 10 of the polymer layer, and therefore it is necessary to form fine pores through the polymer layer in such a manner that they have a pore size greater than that desired for the ultimate membrane filter. The detection of microorganisms by using the membrane filter for the detection of microorganisms according to the present invention can be carried out 15 according to the same procedures used in the detection of microorganisms using the conventional membrane filter. More specifically, microorganisms are filtered off through the membrane filter for the detection of microorganisms according to the present invention, the microorganisms trapped on the membrane filter are stained with a fluorescent dye and then they are observed by a fluorescence 20 microscope. The fluorescent staining operation according to the method of the present invention may be carried out using any conventionally used method for staining microorganisms with a fluorescent dye and specific examples thereof include methods each comprising staining microorganisms with, for instance, Escherichia coli-specific fluorescent probe (Vermicon AG) and/or DAPI 25 (4'6diamino-2-phenyl indole). Examples (Example 1) A membrane filter (CycloblackTM available from Whatman Corporation 5 having a diameter of 25 mm and a pore size, 4, of 0.8 p m) was coated with an Sn according to the sputtering technique. This coating step permitted the formation of an Sn layer on the surface of the membrane filter, and the metal-coated membrane filter was found to have an ultimate pore size, 4 , of 0.55 g m which 5 was equal to that of the membrane filter currently used in the test of microorganisms and smaller than the foregoing pore size, 4), of 0.8 u m (see, Fig. 1). Moreover, in a case where a membrane filter (CycloblackTM available from Whatman Corporation) having a pore size, 4, of 0.6 p m was likewise coated with 10 an Sn, the resulting metal-coated membrane filter was found to have an ultimate pore size, 4, of 0.45 u m which was equal to that of the membrane filter currently used in the test of microorganisms. In a case where a membrane filter (CycloblackTM available from Whatman Corporation) having a pore size, 4, of0.4g m was likewise coated with an Sn, the 15 resulting metal-coated membrane filter was found to have an ultimate pore size, 4), of 0.25 p m (see, Fig. 1). (Example 2) The membrane filter obtained in Example 1 by coating a membrane filter (CycloblackTM available from Whatman Corporation having a diameter of 25 mm, 20 and a pore size, 4 , of 0.8 p m) with an Sn was inspected for background fluorescence intensity encountered when observing the same with a fluorescence microscope. The membrane filters used in this test were as follows: CycloblackTM available from Whatman Corporation as Commercially Available Membrane Filter 1; CycloporeTM (black) available from Whatman Corporation as 25 Commercially Available Membrane Filter 2; and CycloporeTM (plain) available from Whatman Corporation as Commercially Available Membrane Filter 3. In addition, the membrane filter according to the present invention was one prepared by coating Commercially Available Membrane Filter 1 with an Sn layer by the ion-sputtering technique. 6 The method of measuring the background fluorescence intensity was as follows: there was fixed, on the observation zone of a fluorescence microscope (DMRXA2 available from Leica Corporation), either one of the coated membrane filters according to the present invention or the membrane filters each free of any 5 coated layer. Respective fluorescent images of these membrane filters were taken through Fluorescent Filter Block A4 (excitation filter: BP360/40; absorption filter: BP470/40); L5 (excitation filter: BP480/40; absorption filter: BP527/30); and Y3 (excitation filter: BP535/50; absorption filter: BP610/75), using a cooled CCD camera (CoolSNAP). The photographing time (exposure time) was set at 60 ms 10 (A4); 160 ms (L5); and 160 ms (Y3). The intensities of the background fluorescence of these respective membrane filters thus photographed were calculated in terms of the averaged luminance per picture element (gradations of 256) for the fluorescent images per image plane (1300 x 1300 picture elements), with the assistance of an image-analysis software: Leica-Qwin. As a result, the 15 highest value was observed for Commercially Available Membrane Filter 3, and accordingly the background fluorescence values were calculated in terms of the values relative to that observed for the Membrane Filter 3, which was defined to be 100. As shown in Fig. 2, these test results clearly indicate that the membrane 20 filter coated with an Sn layer has a significantly low background fluorescence value as compared with those observed for the membrane filters each free of any metal coating layer. (Example 3) Escherichia coli were filtered through the membrane filter prepared in 25 Example 1 obtained by coating a membrane filter (CycloblackTM available from Whatman Corporation having a diameter of 25 mm and a pore size, 4, of 0.8 A m) with an Sn, and then the membrane filter carrying the bacterial cells trapped thereon were cultivated in an STA culture medium at 37 C for 4 hours. Escherichia col trapped on the membrane filter and cultivated for such a short 7 period of time were fixed with ethanol and then stained with a commercially available Escherichia coli-specific fluorescent probe (Vermicon AG). The staining was carried out according to the protocol annexed to the probe. Thereafter, Escherichia coli were dyed with DAPI (4'6diamino-2-phenyl indole), followed by 5 the adhesion of the membrane filter to a slide glass, the sealing of the same with a sealing agent: SlowFadeTM (Molecular Probe), and the subsequent covering of the membrane filter with a cover glass to thus seal the same. This sample was set on the stage of a fluorescence microscope BX60 (available from Olympus Optical Co., Ltd.) and a CCD camera C5810 (available from Hamamatsu Photonics Co., Ltd.) 10 equipped with an objective lens having a magnification of 60 was used to take micrographs. Fig. 3 shows the Escherichia coli stained with an Escherichia coli-specific probe (on the left-hand side) and the Escherichia coli stained with a DAPI (on the right-hand side), both of them being found in the same field. 15 As will be seen from this figure, Escherichia coli were distinctively stained by either of these staining methods and these results clearly indicate that the staining methods are effective. Brief Description of the Drawings 20 [Figure 1] This figure shows observed surface images of a membrane filter according to the present invention obtained using an electron microscope. [Figure 21 This figure shows background fluorescence observed for commercially available membrane filters and the membrane filter according to the present invention. 25 [Figure 3] This figure shows a micrograph showing an Escherichia coi trapped on the membrane filter according to the present invention and subjected to fluorescent staining. 8